Functional Analysis of Drosophila HSP70 Promoter with Different HSE Numbers in Human Cells

The activation of genetic constructs including the Drosophila hsp70 promoter with four and eight HSE sequences in the regulatory region has been described in human cells. The promoter was shown to be induced at lower temperatures compared to the human hsp70 promoter. The promoter activity increased after a 60-min heat shock already at 38°C in human cells. The promoter activation was observed 24 h after heat shock for the constructs with eight HSEs, while those with four HSEs required 48 h. After transplantation of in vitro heat-shocked transfected cells, the promoter activity could be maintained for 3 days with a gradual decline. The promoter activation was confirmed in vivo without preliminary heat shock in mouse ischemic brain foci. Controlled expression of the Gdnf gene under a Drosophila hsp70 promoter was demonstrated. This promoter with four and eight HSE sequences in the regulatory region can be proposed as a regulated promoter in genetic therapeutic systems.     

In Vitro Interaction of Nuclear Proteins with the Promoter of Soybean Heat Shock Gene Gmhsp17.5E

Proteins present in crude nuclear extracts of soybean (Glycine max) plumules were shown to bind in vitro to the 5′ flanking sequences of the soybean heat shock gene Gmhsp17.5E. The specificity of binding activity present in extracts from both control (28°C) and heat shocked (40°C) tissues was demonstrated by reciprocal competition experiments using gel mobility retardation assays. Footprinting experiments using DNase I with crude nuclear extracts indicated that a continuous stretch of 5′ flanking sequences extending from −40 to −153 was protected from digestion in vitro. Nuclear proteins that were partially purified by heparin agarose chromatography were shown to bind specific TATA-proximal sequences containing the heat shock consensus elements (HSEs) (−73 to −49; −107 to −84) and AT-rich motifs (−119 to −153). Other binding sites within AT-rich sequences (−906 to −888, −868 to 863, −859 to 853, and −841 to −830), distal HSE elements (−568 to −532) and a TATA/dyad (−234 to −207) were also identified by DNase I footprinting of TATA-distal probes. DNA binding activities specific for the HSE and AT-rich sequences were present in nuclear extracts from both control and heat shocked tissues. Both types of binding activity were increased after heat shock treatment; HSE binding increased from 1.8- to 2.7-fold, and binding to AT-rich sequences showed an increase from 1.3- to 1.7-fold.     

Heat shock elements are involved in heat shock promoter activation during tobacco seed maturation

The soybean Gmhsp17.3-B heat shock promoter is developmentally regulated in transgenic tobacco, as indicated by the constitutive expression of a -glucuronidase reporter in seeds [16]. In this paper, we show that both the heat shock promoter-driven -glucuronidase activity and the mRNA of the endogenous Nthsp18P gene accumulate coincident with the onset of seed desiccation. Deletions of the soybean Gmhsp17.3-B promoter, encompassing the heat shock element (HSE)-containing regions, revealed a co-localization of sequences responsible for heat induction and developmental expression. Moreover, synthetic HSEs fused to a TATA box sequence had the potential to stimulate the developmental expression of a GUS reporter gene in seeds of transgenic plants.     

Expression of a heat-inducible gene in transfected mosquito cells

The evolutionary conservation of the heat shock response suggests that plasmids containing promoters from Drosophila heat shock protein (hsp) genes will be useful in the development of gene transfer procedures for cell lines representing a variety of insect species. Conditions for induction of endogenous hsp genes and for expression of the chloramphenicol acetyltransferase (CAT) gene regulated by the Drosophila hsp 70 promoter were examined in Aedes albopictus (mosquito) cells. Five hsps, ranging in size from 27,000 to 90,000 D, were induced in A. albopictus cells during incubation at 41°C in medium containing [³⁵S]methionine. Relative synthesis of these proteins at 37 and 41°C indicated that Aedes hsp 66 is homologous to Drosophila hsp 70. Detection of CAT activity in transfected mosquito cells was enhanced 10-fold under heat shock conditions (6 h, 41°C) based on maximal expression of hsp 66, relative to conditions defined for expression of hsp 70 in Drosophila cells. Analysis of the endogenous heat shock response may be essential to the optimal use of plasmids containing the Drosophila hsp 70 promoter with other insect cell types.     

Transcriptional regulation of an hsp70 heat shock gene in the yeast Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae contains three heat-inducible hsp70 genes. We have characterized the promoter region of the hsp70 heat shock gene YG100, that also displays a basal level of expression. Deletion of the distal region of the promoter resulted in an 80% drop in the basal level of expression without affecting expression after heat shock. Progressive-deletion analysis suggested that sequences necessary for heat-inducible expression are more proximal, within 233 base pairs of the initiation region. The promoter region of YG100 contains multiple elements related to the Drosophila melanogaster heat shock element (HSE; CnnGAAnnT TCnnG). Deletion of a proximal promoter region containing one element, HSE2, eliminated most of the heat-inducible expression of YG100. The upstream activation site (UAS) of the yeast cytochrome c gene (CYC1) can be substituted by a single copy of HSE2 plus its adjoining nucleotides (UASHS). This hybrid promoter displayed a substantial level of expression before heat shock, and the level of expression was elevated eightfold by heat shock. YG100 sequences that flank UASHS inhibited basal expression of UASHS in the hybrid promoter but not its heat-inducible expression. This inhibition of basal UASHS activity suggests that negative regulation is involved in modulating expression of this yeast heat shock gene.     

Stage-Specific Regulation of Murine Hsp68 Gene Promoter in Preimplantation Mouse Embryos

In early mouse embryos, the major inducible heat shock gene, hsp68, is spontaneously and transiently activated at the two-cell stage and becomes heat-inducible around blastocyst stage. We have probed mouse embryo's ability to activate the promoter of this gene during preimplantation development by expression analysis of DNA constructs containing a reporter lacZ gene driven by hsp68 (hsp70A1) 5′-regulatory sequences of various length: (i) a full-length promoter (construct phsplacZ); (ii) a heat shock element (HSE)-deleted promoter (pΔ1hsplacZ); and (iii) a minimal, proximal promoter (pΔ2hsplacZ). When analyzed in transfected L-cells, phsplacZ was heat-inducible, while neither pΔ1hsplacZ nor pΔ2hsplacZ was. Developmental activity of the full-length construct was first analyzed after genome integration in transgenic embryos and found to follow endogenous hsp68 expression in terms of spontaneous activation at the 2-cell stage, down-regulation at the 4-cell stage, and acquisition of heat inducibility at the 16/32-cell stage. In transient expression experiments, injected phsplacZ, pΔ1hsplacZ, and pΔ2hsplacZ were expressed at similar levels by 2-cell embryos, independently of construct topology and injection stage. At the 4-cell stage, however, phsplacZ and pΔ1hsplacZ were expressed at similar levels, while pΔ2hsplacZ was inactive. Only phsplacZ became heat-inducible in late morulas. We conclude that in early mouse embryos, developmental activity of episomic hsp68 promoter depends on proximal sequences at the 2-cell stage and on putative enhancer sequences at the 4-cell stage, while HSEs appear dispensable during early cleavage.